Aldehyde dehydrogenase II

ABSTRACT

The present invention concerns a novel aldehyde dehydrogenase having the following physico-chemical properties: a molecular weight of 100,000±10,000 Da which comprises two homologous subunits or a molecular weight of 150,000±15,000 Da which comprises three homologous subunits, each subunit having a molecular weight of 55,000±2,000 Da; dehydrogenase activity on L-sorbosone, D-Glucosone, D-glucose and D-xylose; utilizes as cofactor pyrroloquinoline quinone; has an optimum pH of from 6.5 to 8.0 for the production of vitamin C and an optimum pH of about 9.0 for the production of 2-keto-L-gulonic acid from L-sorbosone; and is inhibited by Co 2+ , Cu 2+ , Fe 3+ , Ni 2+ , Zn 2+ , and monoiodoacetate, is derived from a microorganism belonging to the genus  Gluconobacter.

The present invention concerns a novel enzyme, namely aldehydedehydrogenase (hereinafter referred to as SNDH II), which is responsiblefor both of the conversions, from L-sorbosone to L-ascorbic acid(hereinafter referred to as vitamin C) at neutral pH, and fromL-sorbosone to 2-keto-L-gulonic acid (hereinafter referred to as 2-KGA)at alkaline pH. The present invention also provides a process forproducing said enzyme and a process for producing vitamin C and/or 2-KGAdirectly from aldoses such as L-sorbosone utilizing said enzyme.

Vitamin C is one of very important and indispensable nutrient factor forhuman beings. The metabolic pathways to produce vitamin C have beenwidely studied in various organisms. However, there is no report aboutpurified enzymes relating to the direct conversion of L-sorbosone tovitamin C. Therefore, the enzyme of the present invention is very usefulfor a novel vitamin C production process substitutive for the currentprocess such as the Reichstein method (Helvetica Chimica Acta 17:311(1934)).

The present invention provides a purified aldehyde dehydrogenase havingthe following physico-chemical properties:

-   a) Molecular weight of 100,000±10,000 Da (consisting of two    homologous subunits) or molecular weight of 150,000±15,000 Da    (consisting of three homologous subunits), where each subunit has a    molecular weight of 55,000±2,000 Da);-   b) Substrate specificity: active on aldehyde compounds,-   c) Cofactor: pyrroloquinoline quinone (PQQ),-   d) Optimum pH of from about 6.5 to about 8.0 (for the production of    vitamin C from L-sorbosone) or optimum pH of about 9.0 (for the    production of 2-keto-L-gulonic acid from L-sorbosone),-   e) Inhibitors: Co²⁺, Cu²⁺, Fe³⁺, Ni²⁺, Zn²⁺, and monoiodoacetate.

In one embodiment, the present invention is related to an aldehydedehydrogenase with a molecular weight of 100,000±10,000 Da having thephysico-chemical properties as described above.

In a further embodiment, the present invention is related to an aldehydedehydrogenase with a molecular weight of 150,000±15,000 Da having thephysico-chemical properties as described above.

The source of the SNDH II of the present invention is not critical.Thus, SNDH II of the present invention can be produced, for example, byisolation from a Gluconobacter or another organism capable of producingthe dehydrogenase having the above properties or it can be producedrecombinantly or by chemical synthesis.

Another object of the present invention provides a process for producingthe SNDH II described above, comprising cultivating a microorganismbelonging to the genus Gluconobacter, which is capable of producing thealdehyde dehydrogenase having the above mentioned properties, in anaqueous nutrient medium under aerobic conditions, disrupting the cellsof the microorganism, and isolating and purifying the aldehydedehydrogenase from the cell-free extract of the disrupted cells of themicroorganism.

In one aspect of the present invention, the process for producing SNDHII as described above is carried out by cultivating a microorganismbelonging to the genus Gluconobacter, which is capable of producing thealdehyde dehydrogenase having the above mentioned properties, whereinthe reaction is carried out at a pH of from about 5.5 to about 9.0 andat a temperature of from about 20 to about 50° C., preferably from about20 to about 40° C., most preferably from about 20 to about 30° C. TheSNDH II thus produced is useful for both the production of vitamin C and2-KGA.

A further object of the present invention provides a process forproducing a carboxylic acid and/or its lactone from its correspondingaldose, comprising contacting the aldehyde with the purified SNDH IIhaving the above mentioned properties, or cell-free extract preparedfrom a microorganism belonging to the genus Gluconobacter which iscapable of producing the aldehyde dehydrogenase having the abovementioned properties in the presence of an electron acceptor.

The aldoses as used herein include but are not limited to L-sorbosone,D-glucosone, D-glucose, and D-xylose.

A preferred lactone is vitamin C, a preferred carboxylic acid is 2-KGAand a preferred aldose is L-sorbosone.

In one embodiment, the process for producing a carboxylic acid and/orits lactone from its corresponding aldose comprises contacting thealdehyde with the purified SNDH II having the above mentioned propertiesor with a cell-free extract prepared from a microorganism belonging tothe genus Gluconobacter as defined above, wherein the molecular weightof SNDH II is 100,000±10,000 Da.

In one embodiment, the process for producing a carboxylic acid and/orits lactone from its corresponding aldose comprises contacting thealdehyde with the purified SNDH II having the above mentioned propertiesor with a cell-free extract prepared from a microorganism belonging tothe genus Gluconobacter as defined above, wherein the molecular weightof SNDH II is 150,000±15,000 Da.

In one aspect, the present invention is directed to a process forproducing a carboxylic acid and/or its lactone from its correspondingaldose comprising contacting the aldehyde with the purified SNDH IIhaving the above mentioned properties or with a cell-free extractprepared from a microorganism belonging to the genus Gluconobacter,which is capable of producing the aldehyde dehydrogenase having theabove mentioned properties, wherein the reaction is carried out at a pHof from about 5.5 to about 9.0 and at a temperature of from about 20 toabout 50° C., preferably from about 20 to about 40° C., most preferablyfrom about 20 to about 30° C. In case of the production of vitamin C,the reaction is carried out preferably at a pH of from about 6.5 toabout 8.0 and at a temperature of from about 20 to about 40° C. In caseof the production of 2-KGA, the reaction is carried out preferably at apH of about 9.0 and at a temperature of from about 20 to about 30° C.

The invention also features the use of the purified aldehydedehydrogenase having the above mentioned properties in the process forthe production of a carboxylic acid and/or its lactone from itscorresponding aldose which comprises contacting the aldehyde with saidpurified aldehyde dehydrogenase or cell-free extract prepared from amicroorganism belonging to the genus Gluconobacter which is capable ofproducing said aldehyde dehydrogenase in the presence of an electronacceptor.

The physico-chemical properties of the purified sample of SNDH IIprepared according to the Examples mentioned hereinafter are as follows:

1) Enzyme Activity

SNDH II of the present invention catalyzes the oxidation of L-sorbosoneto vitamin C and/or 2-KGA in the presence of an electron acceptoraccording to the following reaction equation:L-Sorbosone+Electron acceptor→Vitamin C and/or 2-KGA+Reduced electronacceptor

The enzyme does not work with oxygen as an electron acceptor. This wasaffirmed by the failure of the enzyme to convert L-sorbosone to vitaminC and/or 2-KGA using oxygen as a possible electron acceptor.Furthermore, no oxygen consumption was detected in the reaction mixtureas detected with a dissolved oxygen probe. In addition NAD and NADP arenot suitable electron acceptors. However, other conventional electronacceptors can be utilized in conjunction with the enzyme of thisinvention. Preferred electron acceptors are phenazine methosulfate(PMS), 2,6-dichlorophenolindophenol (DCIP), ferricyanide and cytochromec. There is no minimum amount of electron acceptors which must bepresent for at least some of the aldehyde substrate to be converted toits corresponding acid. However, the amount of substrate which can beoxidized depends on the amount of the particular electron acceptor andits electron accepting characteristics.

The enzyme assay was performed as follows:

a) Assay Determining the Enzyme Activity for the Conversion fromL-Sorbosone to Each Product, Vitamin C or 2-KGA

The reaction mixture consisted of 1.0 mM PMS, 25 mM potassium phosphatebuffer (pH 7.0), 1.0 μM PQQ, 1.0 mM CaCl₂, 50 mM L-sorbosone and enzymesolution in a final volume of 100 μl water, said reaction mixture wasprepared just before the assay. The reaction was carried out at 30° C.for 60 minutes unless otherwise stated. The amount of vitamin C, as theindication for enzyme activity, was measured at a wavelength of 264 nmby a high performance liquid chromatography system (HPLC) which wascomposed of a UV detector (TOSOH UV8000; TOSOH Co., Kyobashi 3-2-4,Chuo-ku, Tokyo, Japan), a dualpump (TOSOH CCPE; TOSOH Co.), anintegrator (Shimadzu C-R6A; Shimadzu Co., Kuwahara-cho 1, Nishinokyo,Chukyo-ku, Kyoto, Japan) and a column (YMC-Pack polyamine II; YMC, Inc.,3233 Burnt Mill Drive Wilimington, N.C. 28403, USA). The amount ofproduced 2-KGA, as another indication for enzyme activity, was measuredby HPLC as described above. One unit of the enzyme activity for eachproduction was defined as the amount of the enzyme which produces 1 mgof vitamin C and 2-KGA, respectively, in the reaction mixture.

b) The Photometrical Assay of SNDH II

The reaction mixture consisted of 0.1 mM DCIP, 1.0 mM PMS, 50 mMpotassium phosphate buffer (pH 7.0), 1.0 μM PQQ, 2 to 100 mM substrate(L-sorbosone, D-glucosone, D-glucose, etc.) and enzyme solution in afinal volume of 100 μl water, said reaction mixture was prepared justbefore the assay. The reaction was started at 25° C. with L-sorbosone,and the enzyme activity was measured as the initial reduction rate ofDCIP at 600 nm. One unit of the enzyme activity was defined as theamount of the enzyme catalyzing the reduction of 1 μmol DCIP per minute.The extinction coefficient of DCIP at pH 7.0 was taken as 14.2 mM⁻¹. Areference cuvette contained all the above constituents except forL-sorbosone.

The protein concentration was measured with Protein Assay CBB Solution(Nacalai tesque, Inc. Kyoto, Japan).

2) Substrate Specificity

a) The substrate specificity of the enzyme was determined using the sameenzyme assay method as described under 1b) above with the exception ofusing 100 mM potassium phosphate (pH 7.5) or 100 mM Tris-HCl (pH 9.0) asbuffer. The relative activity of SNDH II for D-glucosone (2 mM),D-glucose (100 mM), and D-xylose (100 mM) was higher than that forL-sorbosone (2 mM) at both pH 7.5 and 9.0. However, the relativeactivity for L-sorbose (100 mM), D-sorbitol (100 mM), andL-gulono-γ-lactone (100 mM) was lower than 1% of that for L-sorbosone atboth pH 7.5 and 9.0. These results are presented in Table 1A. TABLE 1ASubstrate specificity of the purified enzyme Relative activity (%)Substrate pH 7.5 pH 9.0 L-Sorbosone 100 100 D-Glucosone 483 1591D-Glucose 1769 1519 L-Sorbose <1 <1 D-Sorbitol <1 <1 D-Xylose 2123 1323L-Gulono-γ-lactone <1 <1

b) The deduced products of the oxidation of the substrate indicated inTable 1A are shown in Table 1B below. TABLE 1B Substrate ProductL-Sorbosone Vitamin C/2-KGA D-Glucosone D-iso-Ascorbicacid/2-Keto-D-gluconate D-Glucose D-Gluconate D-Xylose D-Xylonic acid3) Optimum pH

The correlation between the reaction rate of SNDH II and pH values ofthe reaction mixture was determined by the same assay method asdescribed under 1a) above, with the exception that various pHs andbuffers in a concentration of 100 mM were used.

The enzyme showed relatively high activity for the production of vitaminC at a pH of from about 6.5 to about 8.0 and high activity for theproduction of 2-KGA at a pH of about 9.0.

4) Effect of Temperature

The effect of temperature for the enzyme reaction was tested by the sameassay method as described under 1a) above, with the exception thatvarious temperatures were used. In both productions of vitamin C and2-KGA, the enzyme reaction was carried out stable up to at least 40° C.

5) Effects of Metal Ions and Inhibitors

The effects of metal ions and inhibitors on the L-sorbosonedehydrogenase activity of the enzyme were examined by measuring theactivity using the same assay method as described under 1b) above. Eachcompound solution was stirred into the basal reaction mixture and thereaction was started with the addition of the enzyme. The results areshown in Table 2. TABLE 2 Effect of inhibitors and metals on theactivity of the purified enzyme Compound Relative activity (%) None100.0 EDTA 97.9 NaN₃ 98.4 Monoiodoacetate 34.7 CaCl₂.2H₂O 94.7CoCl₂.6H₂O 60.8 CuSO₄ <1 Fe2(SO₄)₃.xH₂O 73.2 NiSO₄.6H₂O 82.9 TiCl₄ 95.9ZnCl₂ 64.9 MgCl₂ 88.5

Each compound was added to the reaction mixture at a concentration of1.0 mM, with the exception that the concentrations of EDTA, NaN₃ andmonoiodoacetate were 5.0 mM.

As shown in Table 2, Co²⁺, Cu²⁺, Fe³⁺, Ni²⁺, and Zn²⁺ inhibited theenzyme activity. The addition of 5.0 mM monoiodoacetate stronglyinhibited the enzyme activity.

6) Molecular Weight

The molecular weight of the enzyme was measured with a size exclusiongel column (TSK-gel G3000 SWXL; TOSOH Co., Akasaka 1-7-7, Minato-ku,Tokyo, Japan). The enzyme showed two peaks corresponding to the apparentmolecular weight of about 100,000±10,000 Da and about 150,000±15,000 Daon the chromatography. By analyzing this enzyme on 10%SDS-polyacrylamide gel electrophoresis stained with CBB, it was shownthat the purified enzyme consisted of two to three homologous subunitseach having a molecular weight of about 55,000±2,000 Da. Both thedimeric and trimeric forms of the enzyme are active.

7) Prosthetic Group

The purified SNDH II (0.1 mg) in 50 μl of 100 mM NaH₂PO₄—HCl (pH about1.0) was added by an equal volume of methanol and mixed well. The samplewas centrifuged to remove a precipitate. The resulting supernatant wasused for analysis of the prosthetic group. The absorption spectrum ofthe extract was almost identical with an authentic sample of PQQ(Mitsubishi Gas Chemical, Japan). Its absorbance peaks were found at 251and 348 nm. Furthermore, by HPLC analysis using a reverse phase column(YMC-Pack Pro C18 AS-312; YMC Co., Ltd) at a wavelength of 313 nm, theextract of SNDH II with methanol showed the same retention time as thatof the authentic PQQ.

The detection of heme c of the purified enzyme was attempted by thereduced-minus-oxidized difference spectrum taken by a UV-VIS recordingspectrophotometer (Shimadzu UV-2200; Shimadzu Co.). The enzyme wassuspended in 50 mM potassium phosphate buffer (pH 7.0) at aconcentration of 50 μg/ml and the enzyme of dithionite-reduced form andammonium persulfate-oxidized form were prepared to measure thedifference spectrum. However, the spectrum obtained did not showapparent peaks at a wavelength of between 450 and 650 nm.

These results strongly suggest that the enzyme has PQQ, but has no hemec as prosthetic group.

8) Effect of Substrate Concentration

The velocity of the oxidizing reaction with various concentrations ofL-sorbosone from 1 mM to 8 mM was measured to determine the Km value forL-sorbosone. The Michaelis constants were calculated to be 14.7 mM and20.0 mM at pHs of 7.5 and 9.0, respectively, from the Lineweaver-Burkplot based on the reaction velocity when DCIP was used as the electronacceptor for the reaction.

9) Purification Procedure

The purification of the enzyme is effected by any combination of knownpurification methods, such as ion exchange column chromatography,hydrophobic column chromatography, salting out and dialysis.

The enzyme provided by the present invention can be prepared bycultivating an appropriate microorganism in an aqueous nutrient mediumunder aerobic conditions, disrupting the cells of the microorganism andisolating and purifying the dehydrogenase from the cell-free extract ofthe disrupted cells of the microorganism.

The microorganisms used for the process of the present invention aremicroorganisms belonging to the genus Gluconobacter which are capable ofproducing the dehydrogenase as defined herein before.

A preferred strain is Gluconobacter oxydans. The strain most preferablyused in the present invention is Gluconobacter oxydans DSM 4025, whichwas deposited at the Deutsche Sammlung von Mikroorganismen in Göttingen(Germany), based on the stipulations of the Budapest Treaty, under DSMNo. 4025 on Mar. 17, 1987. The depositor was The Oriental ScientificInstruments Import and Export Corporation for Institute of Microbiology,Academia Sinica, 52 San-Li-He Rd., Beijing, Peoples Republic of China.The effective depositor was said Institute, of which the full address isThe Institute of Microbiology, Academy of Sciences of China, Haidian,Zhongguancun, Beijing 100080, People's Republic of China.

Moreover, a subculture of the strain has also been deposited at theNational Institute of Advanced Industrial Science and Technology (AIST),Japan, also based on the stipulations of the Budapest Treaty, under thedeposit No. Gluconobacter oxydans DSM No. 4025 (FERM BP-3812) on Mar.30, 1992. The depositor is Nippon Roche K.K., 6-1, Shiba 2-chome,Minato-ku, Tokyo, Japan. This subculture is also most preferably used inthe present invention.

Thus, it is an object of the present invention to provide an aldehydedehydrogenase as defined herein before which is derived fromGluconobacter oxydans having the identifying characteristics of thestrain Gluconobacter oxydans DSM No. 4025 (FERM BP-3812), a subcultureor mutant thereof.

Mutants of G. oxydans DSM 4025 (FERM BP-3812) or a microorganismbelonging to the genus Gluconobacter and having identifyingcharacteristics of G. oxydans DSM 4025 (FERM BP-3812) may be obtained bytreating the cells by means of, for instance, ultraviolet or X-rayirradiation, or a chemical mutagen such as nitrogen mustard orN-methyl-n′-nitro-N-nitrosoguanidine.

Any type of microorganism may be used, for instance, resting cells,acetone treated cells, lyophilized cells, immobilized cells and the liketo act directly on the substrate. Any means per se known as a method inconnection with the incubation technique for micro-organisms may beadopted through the use of aeration and agitated submerged fermenters isparticularly preferred. The preferred cell concentration range forcarrying out the reaction is from about 0.01 g of wet cell weight per mlto 0.7 g of wet cell per ml, preferably from 0.03 g of wet cell per mlto 0.5 g of wet cell per ml.

The microorganism “Gluconobacter oxydans” also includes synonyms orbasonyms of such species having the same physico-chemical properties, asdefined by the International Code of Nomenclature of Prokaryotes.

The characteristics of G. oxydans DSM No. 4025 (FERM BP-3812) are asfollows:

-   a) production of 2-KGA from sorbose,-   b) ethanol is oxidized to acetic acid,-   c) D-glucose is oxidized to D-gluconic acid and 2-keto-D-gluconic    acid,-   d) ketogenesis of polyalcohols,-   e) pellicle and ring growth in mannitol broth (24 h cultivation) at    pH 4 and 5, and pellide growth in glucose broth at pH 4.5,-   f) glycerol is not substantially oxidized to dihydroxyacetone,-   g) production of 2-keto-D-glucaric acid from sorbitol and glucaric    acid but not from glucose, fructose, gluconic acid, mannitol or    2-keto-D-gluconic acid,-   h) polymorphic, apparently no flagella,-   i) brown pigment is produced from fructose,-   j) good growth when co-cultured in the presence of Bacillus    megaterium or a cell extract thereof,-   k) streptomycin sensitive.

The microorganism may be cultured in an aqueous medium supplemented withappropriate nutrients under aerobic conditions. The cultivation may beconducted at a pH of from about 4.0 to about 9.0, preferably from about6.0 to about 8.0. The cultivation period varies depending on the pH,temperature and nutrient medium to be used, and is preferably about 1 to5 days. The preferred temperature for carrying out the cultivation isfrom about 13° C. to about 36° C., more preferably from about 18° C. toabout 33° C. A temperature of up to about 50° C. might be also suitablefor the cultivation of the microorganism.

It is usually required that the culture medium contains such nutrientsas assimilable carbon sources, for example glycerol, D-mannitol,D-sorbitol, erythritol, ribitol, xylitol, arabitol, inositol, dulcitol,D-ribose, D-fructose, D-glucose, and sucrose, preferably D-sorbitol,D-mannitol and glycerol; and digestible nitrogen sources such as organicsubstances, for example, peptone, yeast extract, baker's yeast, urea,amino acids, and corn steep liquor. Various inorganic substances mayalso be used as nitrogen sources, for example nitrates and ammoniumsalts. Furthermore, the culture medium usually contains inorganic salts,for example magnesium sulfate, potassium phosphate and calciumcarbonate.

An embodiment for the isolation and purification of SNDH II from themicroorganism after the cultivation is briefly described hereinafter:

-   (1) Cells are harvested from the liquid culture broth by    centrifugation or filtration.-   (2) The harvested cells are washed with water, physiological saline    or a buffer solution having an appropriate pH.-   (3) The washed cells are suspended in the buffer solution and    disrupted by means of a homogenizer, sonicator or French press or by    treatment with lysozyme and the like to give a solution of disrupted    cells.-   (4) SNDH II is isolated and purified from the cell-free extract of    disrupted cells, preferably from the soluble fraction of the    microorganism.

A cell free extract can be obtained from the disrupted cells by anyconventional technique, including but not limited to centrifugation.

SNDH II provided by the present invention is useful as a catalyst forthe production of vitamin C and/or 2-KGA from L-sorbosone. The reactioncan be conducted at a pH of from about 5.5 to about 9.0 for both ofvitamin C production and 2-KGA production in the presence of an electronacceptor, for example DCIP, PMS and the like in a solvent such asphosphate buffer, Tris-buffer and the like. For vitamin C production,the best results are usually achieved if the pH is set at about 6.5 toabout 8.0 and the temperature is set at about 20 to about 40° C. For2-KGA production, the best results are usually achieved if the pH is setat about 9.0 and the temperature is set at about 20 to about 30° C.

The concentration of L-sorbosone in a reaction mixture can varydepending upon other reaction conditions but, in general, is about 0.5to about 50 g/l, most preferably from about 1 to about 30 g/l.

In the reaction, SNDH II may also be used in an immobilized state withan appropriate carrier. Any means of immobilizing enzymes generallyknown in the art may be used. For instance, the enzyme may be bounddirectly to a membrane, granules or the like of a resin having one ormore functional groups, or it may be bound to the resin through bridgingcompounds having one or more functional groups, for exampleglutaraldehyde.

In addition to the above, the cultured cells are also useful for theproduction of carboxylic acids and/or its lactones from theircorresponding aldoses, especially for the production of 2-KGA and/orvitamin C from L-sorbosone. The production of other carboxylic acidsand/or its lactones from their corresponding aldoses is carried outunder the same conditions, including substrate concentration, as theconversion of L-sorbosone to 2-KGA and/or vitamin C as described above.

The following Examples further illustrate the present invention.

EXAMPLE 1 Preparation of SNDH II

All the operations were performed at 8° C., and the buffer was 0.05 Mpotassium phosphate (pH 7.0) unless otherwise stated.

(1) Cultivation of Gluconobacter oxydans DSM No. 4025 (FERM BP-3812)

Gluconobacter oxydans DSM No. 4025 (FERM BP-3812) was grown on an agarplate containing 5.0% D-mannitol, 0.25% MgSO₄.7H₂O, 1.75% corn steepliquor, 5.0% baker's yeast, 0.5% urea, 0.5% CaCO₃ and 2.0% agar at 27°C. for 4 days. One loopful of the cells was inoculated into 50 ml of aseed culture medium containing 2% L-sorbose, 0.2% yeast extract, 0.05%glycerol, 0.25% MgSO₄.7H₂O, 1.75% corn steep liquor, 0.5% urea and 1.5%CaCO₃ in a 500 ml Erlenmeyer flask, and cultivated at 30° C. with 180rpm for one day on a rotary shaker. The seed culture thus prepared wasused for inoculating 2 liters of medium, which contained 8.0% L-sorbose,0.05% glycerol, 0.25% MgSO₄.7H₂O, 3.0% corn steep liquor, 0.4% yeastextract and 0.15% antifoam, in a 3-1 jar fermentor. The fermentationparameters were 800 rpm for the agitation speed and 0.5 vvm (volume ofair/volume of medium/minute) for aeration at a temperature of 30° C. ThepH was maintained at 7.0 with sodium hydroxide during the fermentation.After 48 hours of cultivation, 6 liters of the cultivated brothcontaining the cells of Gluconobacter oxydans DSM No. 4025 (FERMBP-3812) by using the three sets of fermentors were harvested bycontinuous centrifugation. The pellets containing the cells wererecovered and suspended in an appropriate volume of saline. After thesuspension was centrifuged at 2,500 rpm (1,000×g), the supernatantcontaining the slightly reddish cells was recovered to remove theinsoluble materials derived from corn steep liquor and yeast extractwhich were ingredients for the medium. The supernatant was thencentrifuged at 8,000 rpm (10,000×g) to obtain the cell pellet. As aresult, 38.4 g of the wet weight of cells of Gluconobacter oxydans DSMNo. 4025 (FERM BP-3812) was obtained from 6 liters of the broth.

(2) Preparation of Cytosol Fraction

A portion (19.2 g) of the cell paste was suspended with 100 ml of thebuffer and passed through a French pressure cell press. Aftercentrifugation to remove intact cells, the supernatant was designated asthe cell-free extract, and the cell-free extract was centrifuged at100,000×g for 60 minutes. The resultant supernatant (112 ml) wasdesignated as the soluble fraction of Gluconobacter oxydans DSM No. 4025(FERM BP-3812). After this fraction was dialyzed against the buffer, 112ml of the dialyzed fraction having the specific activity for producingvitamin C from L-sorbosone of 0.172 unit/mg protein were used for thenext purification step.

(3) Diethylaminoethyl (DEAE)-Cellulose Column Chromatography

The dialysate (112 ml) was put on a column of DEAE-cellulose (WhatmanDE-52, 3×50 cm; Whatman BioSystems Ltd., Springfield MIII, James WhatmanWay, Maidstone, Kent, U.K.) equilibrated with the buffer and washed withthe buffer to elute minor proteins. Then a linear gradient elution withNaCl from 0.28 to 0.58 M in the buffer was carried out. Major enzymeactivity was eluted at 0.36 M NaCl. The active fractions (97.5 ml) werecollected.

(4) DEAE-Sepharose Column Chromatography

A portion (97 ml) of the dialyzed active fraction from the previous stepwas put on a column of DEAE-sepharose CL-6B (Pharmacia, 3.0 by 25 cm)equilibrated with the buffer. After the column was washed with thebuffer containing 0.3 M NaCl, a linear gradient of NaCl from 0.3 to 0.45M was added to the buffer. The active fractions were eluted at NaClconcentrations ranging from 0.44 to 0.47 M. The active fractions (40 ml)were collected and dialyzed against the buffer.

(5) Q-Sepharose Column Chromatography (1^(st) Step)

The dialyzed active fraction (40 ml) was put on a column of Q-sepharose(Pharmacia, 1.5 by 25 cm) equilibrated with the buffer. After the columnwas washed with the buffer containing 0.3 M NaCl, a linear gradient ofNaCl from 0.3 to 0.5 M was added to the buffer. The active fractionswere eluted at NaCl concentrations ranging from 0.44 to 0.46 M.

(6) Q-Sepharose Column Chromatography (2^(nd) Step)

The pooled active fractions (17 ml) from the previous step were dialyzedagainst the buffer. The dialyzed sample (17 ml) was put on a column ofQ-sepharose (Pharmacia, 1.5 by 25 cm) equilibrated with the buffer.After the column was washed with the buffer containing 0.33 M NaCl, alinear gradient of NaCl from 0.33 to 0.48 M was added to the buffer. Theactive fractions were eluted at NaCl concentrations ranging from 0.45 to0.48 M.

(7) Hydrophobic Column Chromatography

The active fraction from the previous step was filtrated by anultrafiltrator (Centriprep-10) to desalt and concentrate. A portion (750μl) of the desalted and concentrated sample (780 μl) was added to theequal volume (750 μl) of the buffer containing 3 M ammonium sulfate (thefinal concentration: 1.5 M). After centrifugation (15,000×g) of thesample, the supernatant was put on a hydrophobic column RESOURCE ISO(Pharmacia, bed volume: 1.0 ml) equilibrated with the buffer containing1.5 M ammonium sulfate. After the column was washed with the buffercontaining 1.5 M ammonium sulfate, the proteins were eluted with thebuffer containing a linear gradient of ammonium sulfate from 1.5 to 0.75M. The activities corresponding to SNDH II were eluted at ammoniumsulfate concentrations ranging from 1.04 to 1.00 M. The active fractionswere dialyzed against the buffer using dialysis cups (Dialysis-cup MWCO8000, Daiichi pure chemicals, Nihonbashi 3-13-5, Chuo-ku, Tokyo, Japan).Afterward, the fractions were gathered and stored at −20° C.

A summary of the purification steps of the enzyme is given in Table 3.TABLE 3 Purification of the aldehyde dehydrogenase from Gluconobacteroxydans DSM No. 4025 (FERM BP-3812) Total Total Specific activityactivity protein (units*/mg Step (units) (mg) protein) Soluble fraction151.4 879.3 0.172 DEAE-Cellulose DE52 173.0 37.73 4.584 DEAE-SepharoseCL-6B 45.07 10.63 4.242 Q-Sepharose (1^(st) step) 23.65 1.462 16.17Q-Sepharose (2^(nd) step) 13.70 0.527 26.03 RESOURCE-ISO 4.84 0.09948.90One unit* of the enzyme was defined as the amount of enzyme whichproduces 1 mg of vitamin C per hour in the reaction mixture described in1a).(8) Purity of the Isolated Enzyme

The purified enzyme (0.039 mg/ml) with a specific activity of 48.9 unitsper mg protein for vitamin C production and a specific activity of 12.3units per mg protein for 2-KGA production was used for the followinganalysis:

The molecular weight of the native enzyme was estimated by highperformance liquid chromatography using a size exclusion gel column (TSKgel G3000 SWXL column, 7.8×300 mm) equilibrated with 0.1 M potassiumphosphate buffer (pH 7.0) containing 0.3 M NaCl at 280 nm and a flowrate of 1.5 ml per minute. Cyanocobalamin (1.35 kDa), myoglobin (17kDa), ovalbumin (44 kDa), γ-globulin (158 kDa) and thyroglobulin (670kDa) were used as molecular weight standards. The purified enzyme showedtwo peaks having the molecular weight of 100,000±10,000 Da and150,000±15,000 Da, respectively.

According to sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), the enzyme showed a subunit with a molecular weight of55,000±2,000 Da. Therefore, the purified enzyme was estimated to consistof two or three homologous subunits.

(9) Identification of the Reaction Product

The reaction mixture containing the purified enzyme (0.39 μg),L-sorbosone (50 mM), PMS (1 mM), CaCl₂ (1 mM) and PQQ (1 μM) wasincubated in 100 μl of the buffer for 1 hour at 30° C. The reactionproducts were analyzed on thin layer chromatography (Silica gel 60F²⁵⁴,MERCK, 64271 Darmstadt, Germany) and HPLC. Two kinds of products,vitamin C and 2-KGA, were obtained from the enzyme reaction. For vitaminC, the sample was assayed by an amino-column (YMC-Pack Polyamine-II,YMC, Inc.) on a HPLC system. For 2-KGA, the sample was assayed by a C-18column (YMC-Pack Pro C18, YMC, Inc.) on a HPLC system.

EXAMPLE 2 Effect of pH on the Production of Vitamin C or 2-KGA fromL-Sorbosone by SNDH II

The effect of pH for the enzyme reaction was tested. The reactionmixture containing the purified enzyme (273 ng), L-sorbosone (50 mM),PMS (1 mM), CaCl₂ (1 mM) and PQQ (1 μM) in 100 μl of the buffer (100 mM)was incubated for 1 hour at 30° C. The reaction products were analyzedby HPLC. The result is shown in Table 4. TABLE 4 Effect of pH on theproduction of vitamin C or 2-KGA from L-sorbosone by SNDH II Vitamin Cproduced 2-KGA produced Buffer pH (mg/l) (mg/l) Citrate-NaOH 4.50 0.00.0 Citrate-NaOH 5.50 3.8 27.7 Citrate-NaOH 6.50 64.7 21.2 Potassiumphosphate 6.76 7.8 not done Potassium phosphate 7.15 64.4 not donePotassium phosphate 7.55 76.3 1.0 Potassium phosphate 7.97 49.6 19.5Tris-HCl 7.86 70.7 117.8 Tris-HCl 8.34 18.5 124.0 Tris-HCl 8.83 7.2170.7

EXAMPLE 3 Effect of Temperature on the Production of Vitamin C or 2-KGAfrom L-Sorbosone by SNDH II

The effect of temperature on the enzyme activity was tested. Thereaction mixture containing the purified enzyme (390 ng), L-sorbosone(50 mM), PMS (1 mM), CaCl₂ (1 mM) and PQQ (1 μM) in 100 μl of 25 mMpotassium phosphate buffer (pH 7.0) was incubated for 1 hour at varioustemperatures (20-60° C.). The reaction products were analyzed by HPLC.The result is shown in Table 5. TABLE 5 Effect of temperature on theproduction of vitamin C or 2-KGA from L-sorbosone by SNDH II TemperatureVitamin C produced 2-KGA produced (° C.) (mg/l) (mg/l) 20 187.4 50.6 25218.8 53.5 30 190.7 48.1 35 196.4 40.3 40 176.7 37.2 50 138.0 32.8 6047.3 4.3

1. A purified aldehyde dehydrogenase having the followingphysico-chemical properties: a) Molecular weight of 100,000±10,000 Da(consisting of two homologous subunits) or molecular weight of150,000±15,000 Da (consisting of three homologous subunits), where eachsubunit has a molecular weight of 55,000±2,000 Da); b) Substratespecificity: active on L-sorbosone, D-glucosone, D-glucose, D-xylose; c)Cofactor: pyrroloquinoline quinone (PQQ), d) Optimum pH of from about6.5 to about 8.0 (for the production of vitamin C from L-sorbosone) oroptimum pH of about 9.0 (for the production of 2-keto-L-gulonic acidfrom L-sorbosone), e) Inhibitors: Co²⁺, Cu²⁺, Fe³⁺, Ni²⁺, Zn²⁺, andmonoiodoacetate.
 2. The aldehyde dehydrogenase according to claim 1,which is derived from a microorganism belonging to the genusGluconobacter which is capable of producing said aldehyde dehydrogenase.3. The aldehyde dehydrogenase according to claim 2, wherein themicroorganism is Gluconobacter oxydans having the identifyingcharacteristics of the strain Gluconobacter oxydans DSM No. 4025 (FERMBP-3812), a subculture or mutant thereof.
 4. The aldehyde dehydrogenaseaccording to claim 3, wherein the microorganism is Gluconobacter oxydansDSM No. 4025 (FERM BP-3812), a subculture or mutant thereof.
 5. Aprocess for producing an aldehyde dehydrogenase having the followingphysico-chemical properties: a) Molecular weight of 100,000±10,000 Da(consisting of two homologous subunits) or molecular weight of150,000±15,000 Da (consisting of three homologous subunits), where eachsubunit has a molecular weight of 55,000±2,000 Da); b) Substratespecificity: active on aldehyde compounds, c) Cofactor: pyrroloquinolinequinone (PQQ), d) Optimum pH of from about 6.5 to about 8.0 (for theproduction of vitamin C from L-sorbosone) or optimum pH of about 9.0(for the production of 2-keto-L-gulonic acid from L-sorbosone), e)Inhibitors: Co²⁺, Cu²⁺, Fe³⁺, Ni²⁺, Zn²⁺, and monoiodoacetate, whichcomprises cultivating a microorganism belonging to the genusGluconobacter, which is capable of producing the aldehyde dehydrogenasehaving the above properties, in an aqueous nutrient medium under aerobicconditions, disrupting the cells of the microorganism, and isolating andpurifying the aldehyde dehydrogenase from the cell-free extract of thedisrupted cells of the microorganism.
 6. The process according to claim5, wherein the reaction is carried out at a pH of from about 5.5 to 9.0and at a temperature of from about 20 to about 50° C.
 7. A process forproducing a carboxylic acid and/or its lactone from its correspondingaldose which comprises contacting the aldehyde with the purifiedaldehyde dehydrogenase having the following physico-chemical properties:a) Molecular weight of 100,000±10,000 Da (consisting of two homologoussubunits) or molecular weight of 150,000±15,000 Da (consisting of threehomologous subunits), where each subunit has a molecular weight of55,000±2,000 Da); b) Substrate specificity: active on aldehydecompounds, c) Cofactor: pyrroloquinoline quinone (PQQ), d) Optimum pH offrom about 6.5 to about 8.0 (for the production of vitamin C fromL-sorbosone) or optimum pH of about 9.0 (for the production of2-keto-L-gulonic acid from L-sorbosone), e) Inhibitors: Co, Cu²⁺, Fe³⁺,Ni²⁺, Zn²⁺, and monoiodoacetate, or cell-free extract prepared from amicroorganism belonging to the genus Gluconobacter which is capable ofproducing the aldehyde dehydrogenase having the above properties in thepresence of an electron acceptor.
 8. The process according to claim 5,wherein the microorganism is Gluconobacter oxydans having theidentifying characteristics of the strain Gluconobacter oxydans DSM No.4025 (FERM BP-3812), a subculture or mutant thereof.
 9. The processaccording to claim 8, wherein the microorganism is Gluconobacter oxydansDSM No. 4025 (FERM BP-3812), a subculture or mutant thereof.
 10. Theprocess of claim 7, wherein the lactone is vitamin C, the carboxylicacid is 2-keto-L-gulonic acid and the aldose is L-sorbosone.
 11. Theprocess according to claim 7, wherein the reaction is carried out at apH of from about 5.5 to about 9.0 and at a temperature of from about 20to about 50° C. for the production of vitamin C and 2-keto-L-gulonicacid, respectively.
 12. The process according to claim 7, wherein thereaction is carried out at a pH of from about 6.5 to about 8.0 and atemperature of from about 20 to about 40° C. for the production ofvitamin C, and at a pH of about 9.0 and a temperature of from about 20to about 30° C. for the production of 2-keto-L-gulonic acid.
 13. Aprocess for the production of a carboxylic acid and/or its lactone fromits corresponding aldose which comprises contacting the aldehyde withthe purified aldehyde dehydrogenase of claim 1 or cell-free extractprepared from a microorganism belonging to the genus Gluconobacter whichis capable of producing said aldehyde dehydrogenase in the presence ofan electron acceptor.
 14. The process according to claim 6, wherein themicroorganism is Gluconobacter oxydans having the identifyingcharacteristics of the strain Gluconobacter oxydans DSM No. 4025 (FERMBP-3812), a subculture or mutant thereof.
 15. The process according toclaim 7, wherein the microorganism is Gluconobacter oxydans having theidentifying characteristics of the strain Gluconobacter oxydans DSM No.4025 (FERM BP-3812), a subculture or mutant thereof.
 16. The processaccording to claim 8, wherein the reaction is carried out at a pH offrom about 5.5 to about 9.0 and at a temperature of from about 20 toabout 50° C. for the production of vitamin C and 2-keto-L-gulonic acid,respectively.
 17. The process according to claim 9, wherein the reactionis carried out at a pH of from about 5.5 to about 9.0 and at atemperature of from about 20 to about 50° C. for the production ofvitamin C and 2-keto-L-gulonic acid, respectively.
 18. The processaccording to claim 10, wherein the reaction is carried out at a pH offrom about 5.5 to about 9.0 and at a temperature of from about 20 toabout 50° C. for the production of vitamin C and 2-keto-L-gulonic acid,respectively.
 19. The process according to claim 8, wherein the reactionis carried out at a pH of from about 6.5 to about 8.0 and a temperatureof from about 20 to about 40° C. for the production of vitamin C, and ata pH of about 9.0 and a temperature of from about 20 to about 30° C. forthe production of 2-keto-L-gulonic acid.
 20. The process according toclaim 9, wherein the reaction is carried out at a pH of from about 6.5to about 8.0 and a temperature of from about 20 to about 40° C. for theproduction of vitamin C, and at a pH of about 9.0 and a temperature offrom about 20 to about 30° C. for the production of 2-keto-L-gulonicacid.
 21. The process according to claim 10, wherein the reaction iscarried out at a pH of from about 6.5 to about 8.0 and a temperature offrom about 20 to about 40° C. for the production of vitamin C, and at apH of about 9.0 and a temperature of from about 20 to about 30° C. forthe production of 2-keto-L-gulonic acid.
 22. The process according toclaim 11, wherein the reaction is carried out at a pH of from about 6.5to about 8.0 and a temperature of from about 20 to about 40° C. for theproduction of vitamin C, and at a pH of about 9.0 and a temperature offrom about 20 to about 30° C. for the production of 2-keto-L-gulonicacid.